S. aureus is a gram-positive bacterium grouped within Bacillus sp. on the basis of ribosomal RNA sequences. This immobile coccus grows in aerobic and anaerobic conditions, in which it forms grape-like clusters. Its main habitats are the nasal membranes and skin of warm-blooded animals, in r whom it causes a range of infections from mild to serve, such as pneumonia, sepsis, osteomyelitis, and infectious endocarditis. The organism produces many toxins and is highly effective at overcoming antibiotic effectiveness. In fact, S. aureus is one of the major causes of community-acquired and hospital-acquired infections, and its toxins include super-antigens that cause unique disease entities such as toxic shock syndrome and Staphylococcus-associated scarlet fever. In 1961 it was first reported that this bacteria developed resistance to methicillin, invalidating almost all antibiotics including the most potent beta lactams.
In this regard, reports of bacterial strains becoming resistant to known antibiotics are becoming more common, signaling that new antibiotics are needed to combat all bacterial infections, and particularly combat S. aureus, an organism responsible for many nosocomial infections. Unfortunately, historically the identification of new antibiotics has been painstakingly laborious with no guarantee of success. Traditional methods have involved blindly and randomly testing potential drug candidate molecules, with the hope that one might be effective. Presently, the average cost to discover and develop a new drug is nearly $500 million, and average time for drug development is 15 years from laboratory to patient. Clearly new identification and screening methods that will shorten and reduce the cost of this process are needed.
A newly emerging regime for identifying new antibacterial agents is to first identify gene sequences and proteins required for bacterial proliferation of (“essential genes and essential proteins”) and then conduct a biochemical and structural analysis of the particular target gene or protein in order to identify compounds that interact with the target. Such methodology combines molecular modeling technology, combinational chemistry and the means to design candidate drugs, and affords a more directed alternative to merely screening random compounds with the hope that one might be effective for inhibiting or eradicating a particular bacteria.
Nevertheless, even this preferred approach presents obstacles including the identification of essential genes and proteins, and the design of new assays for the genes thus identified in order to efficiently screen candidate compounds. With report to this approach, several groups have proposed systems for the identification of essential genes. For instance, Zyskind and colleagues propose a method of identifying essential genes in Escherichia coli by subcloning a library of E. coli nucleic acid sequences into an inducible expression vector, introducing the vectors into a population of E. coli cells, isolating those vectors that, upon activation and expression, negatively impact the growth of the E. coli cell, and characterizing the nucleic acid sequences and open reading frames contained on the subclones identified. See WO 00/44906, herein incorporated by reference. The disadvantage of this method is that the overexpression of nonessential genes can also negatively impact the cell, particularly the overexpression of membrane proteins and sugar transport proteins that are not necessary for growth where alternative carbon sources exist. Such proteins typically become trapped in membrane export systems when the cell is overloaded, and would be identified by this methodology. See Muller, FEMS Microbiol. Lett. 1999 Jul. 1; 176(1):219-27.
Another group proposes the identification of growth conditional mutants, and more specifically temperature sensitive (ts) mutants, as a means to identify essential genes in Staphylococcus aureus. See Benton et al., U.S. Pat. No. 6,037,123, issued Mar. 14, 2000, herein incorporated by reference. Each gene is identified by isolating recombinant bacteria derived from growth conditional mutant strains, i.e., following introduction of a vector containing a library of nucleic acid sequences, which would grow under non-permissive conditions but which were not revertants. These recombinant bacteria were found to contain DNA inserts that encoded wild type gene products that replaced the function of the mutated gene under non-permissive growth conditions. By this method, Benton and colleagues were able to identify 38 loci on the S. aureus chromosome, each consisting of at least one essential gene.
The disadvantages of this method are first, the chemical employed to induce mutagenesis (diethyl sulfate, DES) is capable of causing several mutations in the same cell, thereby complicating interpretation of the results. Second, the method is particularly labor intensive in that one must painstakingly analyze replica plates of individual colonies grown at permissive and non-permissive temperatures, where replica plates include both mutant and non-mutant cells. Thus, employing the appropriate level of mutagen to achieve a balance between minimizing the number of non-mutant colonies one must screen in order to identify one mutant, while at the same time avoiding multiple mutations in the same cell, may be an arduous task.
Another group has proposed a transposon mutagenesis system for identifying essential genes called “GAMBIT” (“genomic analysis and mapping by in vitro transposition”), and has used the system to identify essential genes first in the gram positive bacteria Haemophilus influenzae and Streptococcus pneumoniae, and more recently in Pseudomonas aeruginosa. See Akerley et al., Systematic identification of essential genes by In vitro mariner mutagenesis, Proc. Natl. Acad. Sci USA 95(15): 8927-32; Wong and Mekalanos, 2000, Proc. Natl. Acad. Sci. USA 97(18): 10191-96; and Mekalanos et al., U.S. Pat. No. 6,207,384, issued Mar. 27, 2001, herein incorporated by reference. GAMBIT involves first isolating and purifying specific genomic segments of approximately 10 kilobases using extended-length PCR, and creating a high density transposon insertion map of the isolated region using Himar1 transposon mutagenesis. The transposon insertions are then transferred to the chromosome following transformation of the bacteria with the transposon containing vectors, and selection for the antibiotic resistance marker on the transposon. The position of each transposon insertion with respect to a given PCR primer is then determined by genetic footprinting, i.e., by amplifying sub-PCR products using one of the original PCR primers and a primer that recognizes an internal site in the Himar1 transposon. By analyzing the length of PCR fragments thus identified, it is possible to identify regions that are devoid of transposon insertions, thereby signaling regions that might contain essential genes.
While the GAMBIT method is a good technique for looking at a small region of the genome for essential genes, it would be extremely labor intensive to use this method for analyzing the entire genome. Furthermore, GAMBIT is not readily applicable for use in organisms that are less recombinogenic than H. influenzae. 
Another group at Abbott Laboratories has proposed a genome scanning method for identification of putative essential genes in H. influenzae, whereby random transposon insertions are mapped and analyzed to identify open reading frames containing no insertion in order to identify putative essential genes. Reich et al., 1999, Genome Scanning in Haemophilus influenzae for Identification of Essential Genes, J. Bacteriol. 181(16): 4961-68. However, even though transposon insertions were isolated that spanned the whole genome, the authors employed a genomic footprinting technique similar to that used in GAMBIT to map insertions in a short contiguous region of the chromosome. The method further employs the methods of mutation exclusion and zero time analysis in order to monitor the fate of individual insertions after transformation in growing culture, which looks at individual insertions on a case-by-case basis.
Wong and Mekalanos also proposed identifying essential genes in P. aeruginosa by starting with the knowledge of three essential genes in H. influenzae and using genetic footprint analysis to determine if the homologues of these genes are essential in P. aeruginosa. Of three homologues tested, only one was unable to accommodate a transposon insertion. See Wong and Mekalanos, supra. Such results underscore the fact that a gene that is shown to be essential in one species will not necessarily be essential in another, given that some gene products may fulfill different functional roles in different species.
Because of the fact that S. aureus is a major cause of life-threatening infection, and its notorious resistance to antibiotics, various groups have reported approaches for identification of S. aureus essential genes as these genes are useful potential targets for antibacterial chemotherapy and for producing therapeutic and prophylactic vaccines.
The availability of the genome sequence of S. aureus, and related bacteria, makes possible studies attempting to identify genes that are essential for viability of the microorganism in vitro or for its ability to cause infection. The products of both types of genes are potential targets in the effort to produce effective antimicrobial agents. Related thereto, Kuroda et al. recently published in the Lancet the whole genome sequence of two related S. aureus strains (N315 and Mu50) by shot-gun random sequencing. N315 is a meticillin-resistant S. aureus strain isolated in 1982 and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. In their paper Kuroda et al. reported the identification of open reading frames by the use of GAMBLER and GLIMMER programs, and annotation of each by BLAST homology search, motif analysis and protein localisation prediction.
Also, Ji et al. recently reported a method for the identification of essential Staphylococcus genes using conditional phenotypes generated by antisense RNA. (Ji et al., Science, 293: 2266-2269 (Sep. 21, 2001)). Using this method, Ji et al. reported the identification of more than 150 putative essential Staphylococcus genes where antisense ablation was lethal or had growth inhibitory effects. Of these genes, 40% are reportedly orthologs or homologs of known essential bacterial genes.
Further, Xia et. al. recently reported a method reportedly useful for rapid identification of essential genes of Staphylococcus aureus using a vector host-dependent for autonomous replication, PSA3182. This approach is based on the insertion by a single crossover of a specific DNA sequence both in the middle of a structural gene, with the inherent inactivation of the gene, and at its 3′ end, where the insertion does not affect the structural gene but might have a polar effect on downstream genes (Xia et al., Plasmid 42:144-49(1999)). Their approach includes comparison of the frequency of the insertion at these two locations as a means for predicting of the essential character of a particular gene. Accordingly, in their strategy, for each studied gene, different fragments located either in the middle of a coding sequence or at its 3′ end, are introduced into a vector host dependent for autonomous replication, PSA3182. Xia et al., report the use of their approach to test the essential character of four S. aureus genes, nusG, divIB, dbpA and dbpB.
Also, Jana et al., also recently reported a method for identifying genes that are essential in S. aureus, by fusing the gene of interest to an IPTG controllable spac promoter and provide a general approach by constructing a plasmid in which the Cat-Pspac cos sites is flanked by cloning sites suitable for inserting DNA fragments of interest (Jana et al., Plasmid 44:100-4 (2000)).
Still further, Zhang et al., report a method for identifying essential genes of S. aureus using a chromosomally-integrated spac system in combination with a Lac 1-expressing plasmid PFF 40. This combination reportedly provides an inducible, titratable and well-regulated system for testing the requirements of specific gene products for cell viability and conditional lethal phenotypes in S. aureus. (Zhang et al. Gene 235: 297-305 (2000)).
Another method for the identification of bacterial essential genes is entitled Transposon Mediated Differential Hybridisation (TMDH), which is disclosed in WO 01/07651, herein incorporated by reference. This method entails (i) providing a library of transposon mutants of the target organism; (ii) isolating polynucleotide sequences from the library which flank inserted transposons; (iii) hybridising said polynucleotide sequences with a polynucleotide library from said organism; and (iv) identifying a polynucleotide in the polynucleotide library to which said polynucleotide sequences do not hybridise in order to identify an essential gene of the organism. However, the problem with this methodology is that it has a high propensity to lead to false positives, and many essential genes will be missed. Furthermore, the method does not yield any detailed information regarding the loci disrupted by transposons, or whether they were hit more than once.
Previous attempts to generate random tranposon insertions in the S. aureus genome have encountered numerous difficulties. For instance, previous transposon systems for S. aureus have created insertions predominantly concentrated in genomic “hot spots”. In addition, difficulties have been encountered in obtaining viable S. aureus bacteria after electroporation procedures, making it difficult to generate a statistically significant number of mutations for mapping and to differentiate between essential and nonessential mutations.
Thus, there is a great need for more efficient methods to identify essential genes, particularly in S. aureus so that new antibacterial agents may be designed therefrom for use in treatment of S. aureus infections.